PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is both a commensal of humans and a highly dangerous bacterial pathogen. S. aureus pathogenesis is mediated by a large repertoire of secreted and cell wall-associated virulence factors, including a number of potent cytolytic peptides called phenol soluble modulins (PSMs). PSMs are amphipathic, alpha helical peptides that vary in size depending on their classification. The ? type PSMs are ~22 amino acids in size and have been the focus of intense study in recent years. They have been implicated in contributing to the high virulence potential of community-acquired methicillin resistant S. aureus (CA-MRSA) strains, in particular those of the USA300 lineage. While the role of ?PSMs in S. aureus infection has been extensively investigated, significant gaps still exist in our understanding of how they are produced in the bacterial cell. There are five ?PSM peptides produced by most S. aureus strains (PSM?1-4 and the ?-toxin). PSM?1-4 are encoded within the same polycistronic transcript (the ?PSM transcript), yet studies have shown that the relative levels of the four peptides vary considerably. PSM?4 (located at the 3' end of the transcript) is commonly the most abundant, while PSM?3 (the most potent of the four peptides) is typically the least abundant. This variation in ?PSM abundance is suggestive of post-transcriptional regulation. The long-term objective of this project is to understand the molecular mechanism(s) that contribute to ?PSM production in S. aureus. In this proposal, we will specifically investigate the contribution of the small RNA Teg41 to ?PSM production and virulence. Preliminary studies show that Teg41 positively influences ?PSM production at the post-transcription level. We will investigate (i) which of the PSM?1-4 peptides is/are regulated by Teg41, (ii) if Teg41-mediated regulation is facilitated by direct base pairing with the ?PSM transcript, and (iii) at what stage in ?PSM production, and how, Teg41 exerts its influence. To investigate these three aims, we will use a combination of in vitro, in vivo, genetic, biochemical, and molecular biology approaches. We will also utilize a number of cutting edge techniques based on high throughput DNA sequencing (SHAPE-seq, miR-CATCH, and Ribo-seq). The results from this study could have direct implications for human health. Disrupting Teg41-mediated ?PSM-production could dramatically lower the virulence potential of S. aureus and therefore may represent a novel target for therapeutic intervention.